U.S. patent application number 16/174924 was filed with the patent office on 2019-02-28 for integrated epr nmr with frequency agile gyrotron.
The applicant listed for this patent is WASHINGTON UNIVERSITY. Invention is credited to Alexander B. Barnes.
Application Number | 20190064088 16/174924 |
Document ID | / |
Family ID | 54480539 |
Filed Date | 2019-02-28 |
View All Diagrams
United States Patent
Application |
20190064088 |
Kind Code |
A1 |
Barnes; Alexander B. |
February 28, 2019 |
INTEGRATED EPR NMR WITH FREQUENCY AGILE GYROTRON
Abstract
A frequency agile gyrotron for use in combination with an NMR
system is disclosed. The frequency agile gyrotron combined with
EPR-NMR magic angle spinning resonators and cryogenic sample
cooling may increase the sensitivity of solid state NMR with
DNP.
Inventors: |
Barnes; Alexander B.; (St.
Louis, MO) |
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Applicant: |
Name |
City |
State |
Country |
Type |
WASHINGTON UNIVERSITY |
St. Louis |
MO |
US |
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|
Family ID: |
54480539 |
Appl. No.: |
16/174924 |
Filed: |
October 30, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15310509 |
Nov 11, 2016 |
10113984 |
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PCT/US2015/030333 |
May 12, 2015 |
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16174924 |
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61993595 |
May 15, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01R 33/60 20130101;
G01R 33/345 20130101; G01R 33/307 20130101; G01R 33/62 20130101;
G01N 24/12 20130101; G01R 33/31 20130101; G01R 33/282 20130101 |
International
Class: |
G01N 24/12 20060101
G01N024/12; G01R 33/30 20060101 G01R033/30; G01R 33/60 20060101
G01R033/60; G01R 33/28 20060101 G01R033/28; G01R 33/62 20060101
G01R033/62 |
Claims
1. A frequency agile gyrotron system for DNP NMR comprising: an NMR
spectrometer; a signal processor operatively connected to the NMR
spectrometer, wherein the signal processor receives one or more
voltages from the NMR spectrometer and produces a control signal;
and a frequency agile gyrotron operatively coupled to the NMR
spectrometer and to the signal processor, the frequency agile
gyrotron configured to emit a broad-banded microwave output
comprising a gyrotron bandwidth; wherein: the NMR spectrometer
controls a frequency of the broad-banded microwave output via the
control signal, the frequency agile gyrotron responds to the
control signal on a timescale of microseconds, and the gyrotron
bandwidth is wider than an EPR linewidth and an NMR frequency.
2. The system of claim 1, wherein the bandwidth of the frequency
agile gyrotron is between about 10 MHz and about 1000 MHz.
3. The system of claim 1, wherein: the NMR spectrometer further
comprises a magnetron injection gun comprising a cathode and an
anode; and the one or more voltages from the NMR spectrometer are
chosen from at least one of: a cathode voltage, an anode voltage,
and an acceleration voltage comprising a voltage difference between
the cathode voltage and the anode voltage.
4. The system of claim 1, wherein the frequency agile gyrotron is
operated as a backward wave oscillator.
5. The system of claim 1, wherein the frequency agile gyrotron
produces the broad-banded microwave output at a phase and frequency
stable condition.
6. The system of claim 5, wherein the broad-banded microwave output
is sliced or gated to provide at least one of: a wide instantaneous
bandwidth comprising short pulses on a nanosecond scale and an
adjustable power transmission length for phase control.
7. The system of claim 1, wherein the NMR spectrometer further
comprises a combined EPR-NMR magic angle spinning resonator.
8. The system of claim 1, further comprising a helium cooling
system for cooling a sample to below about 5 to about 60 Kelvin
with helium using a spinning MAS rotor as a centrifugal gas
compressor.
9. A method of DNP NMR using a frequency agile gyrotron system
comprising an NMR spectrometer operatively coupled to a frequency
agile gyrotron, the method comprising controlling an output
frequency of a broad-banded microwave output produced by the
frequency agile gyrotron by changing an operational voltage of the
frequency agile gyrotron in response to a control signal
corresponding to at least one voltage received from a magnetron
injection gun of the NMR spectrometer, the at least one voltage
chosen from: a cathode voltage, an anode voltage, and an
acceleration voltage comprising a voltage difference between the
cathode voltage and the anode voltage.
10. The method of claim 9, wherein controlling the output frequency
of the broad-banded microwave output produced by the frequency
agile gyrotron comprises at least one of: sweeping the output
frequency on a timescale ranging from nanoseconds to microseconds;
producing the broad-banded microwave output in short pulses; and
producing the broad-banded microwave output in a phase and
frequency stable form and gating the broad-banded microwave output
with at least one nanosecond scale switches.
11. The method of claim 9, further comprising at least one of:
performing at least one time-domain DNP transfer; transferring
polarization from electrons to a nucleus using hyperfine couplings
of greater than 10 KHz; decoupling an electron spin from a nuclear
spin; and manipulating EPR spins during magic angle spinning NMR
and EPR experiments to measure EPR to NMR distances and
orientations.
12. The method of claim 11, wherein the operational voltage of the
frequency agile gyrotron is changed on a timescale ranging from
nanoseconds to microseconds to perform the at least one time-domain
DNP transfer.
13. The method of claim 12, wherein the at least one time-domain
DNP transfer is accomplished using at least one transfer mechanism
chosen from: integrated solid effect, a nuclear orientation via
electron spin locking, and an electron nuclear cross
polarization.
14. The method of claim 9, further comprising cooling a sample to
below about 5 to about 60 Kelvin with helium using a spinning MAS
rotor as a centrifugal gas compressor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 15/310,509, filed on Nov. 11, 2016,
which is hereby incorporated by reference herein in its entirety.
U.S. patent application Ser. No. 15/310,509 is a U.S. National
Phase Application of WO Application No. PCT/US2015/030333, filed
Nov. 11, 2016, which is hereby incorporated by reference herein in
its entirety. WO Application No. PCT/US2015/030333 claims the
benefit of priority to U.S. Provisional Application No. 61/993,595,
filed on May 15, 2014, which is hereby incorporated by reference
herein in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to frequency agile gyrotrons
used to improve magnetic resonance experiments.
BACKGROUND
[0003] Dynamic Nuclear Polarization (DNP) has emerged as a powerful
strategy to increase the sensitivity of NMR experiments on a wide
range of biological systems by transferring the large polarization
of electron spins (EPR) to nuclear spins (NMR). Crucial to the
successful implementation of DNP in conjunction with magic angle
spinning (MAS) has been the development of gyrotrons and NMR
probes, instrumentation used to perform DNP. DNP currently enhances
the sensitivity of NMR experiments on membrane proteins by a factor
of about 50 and on model systems up to about 120 at 9 Tesla.
[0004] The larger gyromagnetic ratio of electron spins compared to
proton spins, lower temperatures, and faster recycle delays all
combine to potentially increase the NMR sensitivity by a factor of
360,000. The associated experimental averaging time may decrease by
a factor of 133 billion. Transferring 100% of the polarization from
the electron spins and cooling samples to 5 K to achieve the
theoretical gains poses an ongoing challenge.
[0005] The microwave source (usually a gyrotron) used in
contemporary DNP experiments is left locked on the same frequency
for continuous-wave operation during the entire experiment. This is
because although DNP gyrotrons have high microwave power output
levels (>10 W), they have not yet been tuned on a fast timescale
in existing magnetic resonance experiments.
[0006] Current MAS DNP technology may experience difficulty
achieving sufficient control of EPR spins. Only a fraction of the 1
GHz broad nitroxide lineshape can be covered with a non-tunable 1
MHz .gamma.B1 microwave field of about 200 GHz that exerts control
over the EPR spins. Others have not been able to use EPR spin
labels on peptides for DNP because of extensive paramagnetic
broadening. Therefore, there is a need for a frequency agile
gyrotron microwave source that can output short pulses to not only
sweep-through the EPR linewidth, but also to control all of the EPR
spins simultaneously with a broad excitation bandwidth. At the same
time, there is a need to increase the .gamma.B1 microwave field
strength by about 3 orders of magnitude (from about 1 MHz to about
1 GHz).
SUMMARY
[0007] In various aspects of the disclosure, a frequency agile
gyrotron system for DNP NMR is provided that includes: an NMR
spectrometer; a signal processor operatively connected to the NMR
spectrometer; and a frequency agile gyrotron operatively coupled to
the NMR spectrometer and to the signal processor. The signal
processor receives one or more voltages from the NMR spectrometer
and produces a control signal. The frequency agile gyrotron is
configured to emit a broad-banded microwave output that includes a
gyrotron bandwidth. The NMR spectrometer controls a frequency of
the broad-banded microwave output via the control signal. The
frequency agile gyrotron responds to the control signal on a
timescale of microseconds. The gyrotron bandwidth is wider than an
EPR linewidth and an NMR frequency.
[0008] The bandwidth of the frequency agile gyrotron may be between
about 10 MHz and about 1000 MHz. The NMR spectrometer may further
include a magnetron injection gun that includes a cathode and an
anode. The one or more voltages from the NMR spectrometer are
chosen from at least one of: a cathode voltage, an anode voltage,
and an acceleration voltage comprising a voltage difference between
the cathode voltage and the anode voltage. The frequency agile
gyrotron may be operated as a backward wave oscillator. The
frequency agile gyrotron may produce the broad-banded microwave
output at a phase and frequency stable condition, and the
broad-banded microwave output may be sliced or gated to provide at
least one of: a wide instantaneous bandwidth that includes short
pulses on a nanosecond scale and an adjustable power transmission
length for phase control. The NMR spectrometer may further include
a combined EPR-NMR magic angle spinning resonator. The system may
further include a helium cooling system for cooling a sample to
below about 5 to about 60 Kelvin with helium using a spinning MAS
rotor as a centrifugal gas compressor.
[0009] In another aspect, a method of DNP NMR using a frequency
agile gyrotron system that includes an NMR spectrometer operatively
coupled to a frequency agile gyrotron is provided. The method
includes controlling an output frequency of a broad-banded
microwave output produced by the frequency agile gyrotron by
changing an operational voltage of the frequency agile gyrotron in
response to a control signal corresponding to at least one voltage
received from a magnetron injection gun of the NMR spectrometer.
The at least one voltage may be chosen from: a cathode voltage, an
anode voltage, and an acceleration voltage that is a voltage
difference between the cathode voltage and the anode voltage.
Controlling the output frequency of the broad-banded microwave
output produced by the frequency agile gyrotron may include at
least one of: sweeping the output frequency on a timescale ranging
from nanoseconds to microseconds; producing the broad-banded
microwave output in short pulses; and producing the broad-banded
microwave output in a phase and frequency stable form and gating
the broad-banded microwave output with at least one nanosecond
scale switches. The method may further include at least one of:
performing at least one time-domain DNP transfer; transferring
polarization from electrons to a nucleus using hyperfine couplings
of greater than 10 KHz; decoupling an electron spin from a nuclear
spin; and manipulating EPR spins during magic angle spinning NMR
and EPR experiments to measure EPR to NMR distances and
orientations. The operational voltage of the frequency agile
gyrotron may be changed on a timescale ranging from nanoseconds to
microseconds to perform the at least one time-domain DNP transfer.
The at least one time-domain DNP transfer may be accomplished using
at least one transfer mechanism chosen from: integrated solid
effect, a nuclear orientation via electron spin locking, and an
electron nuclear cross polarization. The method may further include
cooling a sample to below about 5 to about 60 Kelvin with helium
using a spinning MAS rotor as a centrifugal gas compressor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The following figures illustrate various aspects of the
disclosure.
[0011] FIG. 1 is an illustration of a 197 GHz high-power gyrotron
oscillator for frequency-agile DNP.
[0012] FIG. 2A is projected nitroxide and BDPA EPR lineshapes and
DNP enhancement profiles overlaid on projected power vs. frequency
plot (from a 250 GHz gyrotron). Decoupling frequencies are marked
as .omega..sub.DEC and enhancement frequencies are marked as
.omega..sub.DNP. FIG. 2B shows pulse sequence schemes achievable
with a frequency agile gyrotron. Electron-nuclear decoupling and
frequency modulation of the gyrotron output frequency will enable
improvements in DNP performance.
[0013] FIG. 3 is a pulse sequence for electron dephased REDOR.
[0014] FIGS. 4A, 4B, 4C, 4D, 4E, 4F, and 4G are schematic
representations of NMR DNP probe instrumentation.
[0015] FIGS. 5A, 5B, 5C, 5D, 5E, and 5F are modulation schemes of
the anode voltage accomplished with the circuit shown in FIG. 6.
FIG. 5A is a stepped voltage and frequency switching scheme. FIG.
5B is a stepped and sinusoidal modulation scheme superimposed. FIG.
5C is a saw-toothed function scheme. FIG. 5D is a sinusoidal
modulation from an alternating (AC) current radio frequency circuit
scheme. FIG. 5E is a tangential envelope for adiabatic passage
scheme. FIG. 5F is a stochastic voltage and frequency modulation
scheme.
[0016] FIG. 6 is a scheme of a frequency control circuit of a
gyrotron using an integrated NMR EPR spectrometer.
[0017] FIG. 7 is a simulation of a microwave electromagnetic field
of the DNP sample chamber.
[0018] FIG. 8A is a projected nitroxide EPR lineshape and DNP
enhancement profile overlaid on a projected power spectrum of the
tunable gyrotron. FIG. 8B shows a scheme using frequency modulated
MW power (marked DNP) to transfer EPR polarization efficiently to
nuclei followed by adiabatic EPR inversions to measure long-range
electron-nuclear distances and hyperfine decoupling during NMR
signal acquisition. FIG. 8C is a graph of the chaotic operation of
the gyrotron used for hyperfine decoupling. FIG. 8D shows the
expansion of the highlighted time points in FIG. 8C showing
instantaneous microwave bandwidth is much wider than the nitroxide
EPR linewidth.
[0019] FIG. 9A is a graph of strong hyperfine couplings that may
enable long-range distance measurements (top) compared to weak
.sup.13C-.sup.13C couplings (bottom). FIG. 9B shows the dephasing
of rotational Hahn-echoes with rotor synchronized adiabatic EPR
inversions that may yield curves that can be fit to precise
long-range distances. FIG. 9C shows similar curves can be fit to
yield precise .sup.13C-.sup.13C distances, but only out to about 5
.ANG..
[0020] FIGS. 10A, 10B, and 10C are illustrations of a Fabry-Perot
EPR resonator for magic angle spinning DNP.
[0021] FIG. 11 is a schematic of a miniaturization of helium
recirculation for cryogenic MAS DNP that may reduce the cost and
footprint of recycling helium and permit long-term operation at
cryogenic temperatures of less than about 27 K.
[0022] FIGS. 12A and 12B are illustrations of a miniature MAS
rotor-driven helium recirculation system.
[0023] FIGS. 13A, 13B, and 13C illustrate exogenous (FIGS. 13A and
13B) and mounted EPR spins (FIG. 13C). FIG. 13A illustrates
TOTAPOL. FIG. 13B illustrates water soluble BDPA. FIG. 13C
illustrates nitroxide spin labels, TAOC and MTSSL (left) and
chelated gadolinium (right).
[0024] Corresponding reference characters and labels indicate
corresponding elements among the views of the drawings. The
headings used in the figures should not be interpreted to limit the
scope of the claims.
DETAILED DESCRIPTION
[0025] NMR spectroscopy currently does not utilize higher dimension
spectra to yield better resolution primarily because of the
sensitivity required to record each additional dimension. A
frequency agile gyrotron may provide the sensitivity and
instrumentation to overcome these limitations.
[0026] A frequency agile gyrotron (or a backward wave oscillator,
BWO) microwave source that can output short pulses may allow not
only to sweep-through the EPR linewidth, but also to control all of
the EPR spins simultaneously with a broad excitation bandwidth. At
the same time, the .gamma.B1 microwave field strength may be
increased by 3 orders of magnitude (from about 1 MHz to about 1
GHz). Higher power output from the frequency agile gyrotrons and an
EPR resonator with a quality factor of about 100 will yield an
about 1 GHz .gamma.B1 and enhanced control of the 1 GHz broad
nitroxide EPR resonance. To take advantage of the new EPR control,
cryogenic operation may be required to extend electron spin
coherence lifetimes. In an aspect, the sample may be cooled to
about 27 K. The system may include a miniature closed loop helium
cooling apparatus that uses the spinning sample rotor as a
centrifugal helium compressor. Cryogenic and THz technology may be
able to utilize mounted EPR spin labels to simultaneously measure
multiple long-range (50.+-.0.2 .ANG.) electron nuclear
distances.
[0027] Gyrotron oscillators, or backward wave oscillators, can have
a sufficient frequency and phase stability to provide a stable
microwave beam. The beam can then be sliced and manipulated with
already established semiconductor light activated switches to yield
nanosecond scale pulses (GHz scale bandwidth), and also phase
control by means of adjustable power transmission lengths.
[0028] Provided herein is a frequency agile gyrotron system for use
in DNP NMR or combined EPR-NMR. The frequency agile gyrotron system
may include a broadband gyrotron microwave source, combined EPR-NMR
magic angle spinning resonators, and extreme cryogenic sample
cooling to increase the sensitivity of solid state Nuclear Magnetic
Resonance (NMR) experiments by a factor of 20,000 with novel
time-domain Dynamic Nuclear Polarization (DNP). This tremendous
boost in sensitivity and control of EPR spins may result in
acquiring data six orders of magnitude faster than conventional NMR
and may permit multiple simultaneous electron-nuclear distances
measurements out to 50 .ANG.. The applications of this technology
development and structure determination methodology may have
applications to proteins, molecules, and chemical architectures of
structural interest.
[0029] A gyrotron with frequency agility may be tuned by changing
the operating voltage. Although it is possible to change the
gyrotron frequency by changing the operating magnetic field; such a
method is not amenable to fast tuning schemes due to the
significant inductance of the gyrotron magnet. The same magnetic
tuning previously seen in DNP gyrotrons can also be accomplished
with voltage tuning. However, a 460 GHz gyrotron for 700 MHz DNP
experiments does not have enough power (>10 W) over the entire
nitroxide EPR lineshape (.about.1.8 GHz broad at 16.4 Tesla, or 700
MHz 1H). Exert control over all of the electron spins with a strong
microwave field enables enhanced control the DNP Hamiltonian and
improved DNP performance.
[0030] The frequency agile gyrotron may have the ability to change
the voltage and gyrotron frequency on a timescale ranging from
nanoseconds to microseconds, which may improve DNP and magnetic
resonance spectroscopy. Electron decoupling may be used with the
frequency agile gyrotron, which is analogous to proton decoupling.
In addition, the frequency agile gyrotron may enable Electron
Dephased Rotational Echo Double Resonance (ED-REDOR), which is
analogous to classical nuclear spin dephased REDOR. The ability to
control the microwave irradiation frequency of gyrotrons during DNP
may allow significantly more control over the DNP Hamiltonian. As a
result, beneficial interactions may be turned on and detrimental
ones turned off, resulting in significantly improved performance.
DNP can routinely provide substantial sensitivity gains, but there
are still tremendous opportunities for advancements.
Frequency-agile gyrotrons can overcome many of the current
limitations of DNP including: 1) Poor performance at temperatures
higher than 100 K; 2) Inhomogenous line-broadening; 3) Inverse
scaling enhancements with magnetic field; 4) Paramagnetic
broadening; 5) Failure at MAS frequencies >.about.8 KHz; and 6)
Disperse polarization.
Electron-Nuclear Decoupling in DNP
[0031] Dynamic nuclear polarization (DNP) may increase the
sensitivity of NMR experiments on a wide range of biological
systems. The sensitivity of DNP experiments is generated from
transferring the large polarization (sensitivity) in the electron
paramagnetic resonance (EPR) spin reservoir to nuclear spins.
Strong hyperfine couplings yield fast and efficient electron to
nuclear polarization transfer. However, nuclear spins with strong
hyperfine couplings suffer from extensive paramagnetic broadening.
The method of DNP NMR with a frequency agile gyrotron may first
utilize strong hyperfine couplings to transfer polarization, and
then switch on a strong electron-decoupling field. The pulse
sequence in FIG. 8B implements a DNP polarization (.omega.DNP)
period followed by hyperfine decoupling (.omega.Decouple). This
allows close-in nuclei to quickly become polarized, followed by a
period of hyperfine decoupling that permits spectroscopy on the
close-in .sup.13C spins on a protein and any bound ligands.
[0032] Electron spins on stable organic radicals interact with the
magnetic field 657 times stronger than .sup.1H nuclear spins,
resulting in a theoretical maximum gain in sensitivity of a factor
of 657 as illustrated in the equation of polarization (Eqn. (I))
below. The decreasing sensitivity of NMR experiments on
biomedically relevant preparations may be compensated for with
drastic gains in sensitivity provided by transferring polarization
from electron to nuclear spins (DNP), cooling samples to about 27
Kelvin, and repeating experiments faster by utilizing the short
relaxation time of electron spins.
[0033] In biomedically relevant NMR samples, the electron to
nuclear DNP sensitivity transfer works efficiently only at
temperatures below about 100 K. Such cryogenic temperatures also
inherently boost NMR sensitivity by increasing the population of
spins occupying the lower energy level--note that temperature is a
denominator in Eqn. (I). These two enhancement effects are
multiplicative, meaning the experimentally realistic gain in NMR
sensitivity for DNP experiments performed at 27 Kelvin is a factor
of 5000 (or 2.5.times.10.sup.7 in time). Yet another advantage to
the use of DNP in NMR experiments is that the recycle delay between
NMR scans is governed by the relaxation properties of the electron
spins, which is much faster than nuclear spins, and can result in
100 times faster experimental averaging.
[0034] In an aspect, DNP may enhance the sensitivity of NMR
experiments on membrane proteins by a factor of about 50. Electron
nuclear decoupling experiments employed with a frequency agile
gyrotron (see FIG. 1) may improve DNP enhancement factors to 500,
while also reducing the recycle delays from about 3 seconds to 300
milliseconds. Advanced helium cooled NMR probe instrumentation may
permit NMR experiments at temperatures as low as about 27 K,
resulting in a sensitivity gain of about a factor of 20,000 and a
data collection rate about 400 million times faster than
conventional solid state NMR.
[0035] The successful implementation of DNP in conjunction with
magic angle spinning (MAS) for biomolecular structure determination
has been enabled by the development of gyrotrons and NMR probes.
However, gyrotrons that can switch the microwave frequency quickly
have not yet been employed in DNP experiments. By switching the
gyrotron frequency from 197.0 GHz to 197.3 GHz on a timescale of
microseconds, the EPR spins may be irradiated and partially average
out the electron-nuclear dipolar interactions with an about 2 MHz
continuous microwave decoupling field (FIG. 2A). This frequency
jump may be achieved by decreasing the operating voltage of the
gyrotron by about 670 V. Decoupling the electron spins during the
NMR acquisition (FIG. 2B, scheme 1) may improve sensitivity, since
many NMR signals are extinguished due to direct interactions with
the EPR spins (paramagnetic relaxation effects). Decoupling the
electron spins may further have an impact on the NMR spectroscopy
that can be performed on nuclear spins that otherwise suffer from
extensive paramagnetic broadening.
[0036] The strong electron-nuclear dipolar interaction not only
broadens NMR spectra, but also creates a so-called spin diffusion
barrier. This barrier to nuclear polarization dispersion exists
because strong electronuclear dipolar couplings shift the
resonances of the protons close into the polarizing agent too far
in frequency from resonances from "bulk" protons. The spin
diffusion barrier is detrimental to DNP performance for two
reasons. The close-in protons actually drain the polarization from
the electron, hindering that polarization from getting to the bulk
spins. In addition, the very strong electron-nuclear dipolar
couplings cannot be leveraged for DNP. The couplings of up to 7 MHz
yield fast and efficient DNP transfers of polarization from the
electron.
[0037] Those strong couplings may be utilized and in turn permit
DNP at physiological temperatures and higher spinning frequencies,
and also improve DNP enhancements at cryogenic temperatures. The
pulse sequence in FIG. 2B, scheme 2, implements switched
polarization (.omega.DNP) and decoupling (.omega.DEC) periods. This
enables close-in protons to quickly become polarized, followed by a
time when the .omega.DEC field collapses the spin diffusion barrier
to allow the polarization to be efficiently spread to the bulk.
Electron-nuclear decoupling and frequency modulation of the
gyrotron output frequency enables drastic improvements in DNP
performance.
[0038] DNP experiments on membrane proteins have previously used
exogenous biradical EPR polarizing agents. Due to the about 100
.ANG. physical separation between these EPR spins to the nuclear
spins of structural interest, the enhanced EPR polarization must
undergo an inefficient relayed polarization transfer. In an aspect,
the .sup.13C spins may be polarized directly with rigid amide
nitroxide residues incorporated into the protein domains.
[0039] In an aspect, similar to the orientation of the biradicals
in exogenous EPR polarizing agents, rigid peptide amide nitroxides
must have an orthogonal orientation of the two g-tensors. In this
aspect, the 90.degree. orientation of the amide radicals in FIG. 9C
may yield a separation of EPR frequencies that match the .sup.13C
Larmor frequency (75 MHz) and thus polarize nuclear spins
effectively. Solid phase peptide synthesis may allow incorporation
of nitroxide residues and selective labeling of .sup.13C sites that
may be important to ligand binding.
[0040] Typically, resolution is compromised due to uniform .sup.13C
labeling and inhomogenous broadening of cryogenic MAS experiments.
In an aspect, the method may not require uniform .sup.13C labeling.
Isotope labels may only be used on sites that encode important
structural information on ligand binding, such as but not limited
to .sup.13C on Trp252, Leu251, Met239, bryostatin and prostratin.
In FIG. 9A, many of the 27 predicted correlation peaks may be
resolved in the .sup.13C-.sup.13C 2D NMR spectrum. The linewidth (1
ppm) of the predicted resonances in FIG. 9A comes from MAS DNP
spectra found in FIG. 8A. .sup.13C-.sup.13C correlations peaks are
often inhomogenously broadened in DNP spectra due to the cryogenic
trapping of multiple conformations. However, each distinct
conformation is characterized by correlated chemical shifts that
can be exploited to increase spectral resolution with a double
quantum correlation in the indirect .sup.13C dimension. Such
line-narrowing strategies have not yet been employed to structure
determination efforts of membrane proteins with MAS DNP.
Time Domain DNP Transfers with Frequency Swept or Broadband
Gyrotron Oscillators
[0041] A phenomenon referred to as the Cross Effect is active when
the EPR lineshape is wider than the nuclear Zeeman frequency. This
is the case for nitroxide radicals. For example the about 1000 MHz
lineshape of the nitroxide EPR spectrum shown in FIG. 2A is greater
than the corresponding 300 MHz proton frequency. The Cross Effect
can be understood in a cross-relaxation framework. When the
microwave frequency is targeted on the low-frequency side (about
197.3 GHz in FIG. 2A), the microwaves burn a hole in the mostly
inhomogenously broadened EPR spectrum. In other words, the Zeeman
spin states of the electron spins near the irradiation frequency
become nearly equal. When these spins relax back to their
equilibrium Zeeman population, they cross-relax another electron
spin on the other side of the EPR lineshape along with a nuclear
spin. These nuclear spin states than become polarized according to
the Boltzmann distribution of the electron spin states, resulting
in DNP enhancements of the NMR sensitivity.
[0042] The amount of the EPR spectrum that is saturated from the
microwave field is thus an important factor in Cross Effect DNP. If
fewer electron spins are saturated, fewer spins participate in DNP
and the enhancements are smaller. It follows that a strategy that
increases the saturation bandwidth of the microwave field would
lead to higher DNP enhancements. A fast (>10 KHz) frequency
modulation of the gyrotron frequency, with sufficient microwave
power, will accomplish this. Modulating the microwave frequency
over the lower frequency side of the EPR spectrum may (shading in
FIG. 2A) result in more nuclear polarization and NMR
sensitivity.
[0043] Time domain DNP transfers such as the Integrated Solid
Effect (ISE), Nuclear Orientation via Electron Spin Locking
(NOVEL), electron nuclear cross polarization, and other
irradiations schemes have been proven to yield fast, efficient
transfers at low (.about.9 GHz) microwave frequencies. All of these
techniques could be extended to operate at higher frequencies
(100-1000 GHz) with the use of frequency swept gyrotrons (or BWOs),
or frequency and phase stable gyrotrons (or BWOs) that supply a
microwave beam that can be sliced and manipulated with light
activated semiconductors switches. All of these time domain schemes
have the possibility of transferring polarization from electrons to
nuclei fast enough to allow Optical Polarized DNP at high magnetic
fields, and to perform EPR to NMR polarization transfers
efficiently at temperatures >200 Kelvin.
Simultaneous EPR-NMR Distance Measurements up to 50 .ANG.
[0044] A 1/r.sup.3 distance dependence of the dipolar interaction
encodes biomolecular structure (see FIG. 9A). Although homonuclear
dipolar couplings (labelled .sup.13C-.sup.13C in FIG. 9A) can be
determined precisely to measure short-range distances, longer
distances are more challenging to measure due to the weak
nuclear-nuclear dipolar interaction. The electron-nuclear
(hyperfine) interaction is 2600 times stronger due to the large
magnetic moment of the electron spin. Strong hyperfine couplings
may be used to measure electron-nuclear distances on a protein out
to about 50 .ANG..
[0045] FIG. 8A illustrates the projected nitroxide EPR lineshape
and DNP enhancement profiles overlaid on the projected power
spectrum of the frequency agile gyrotron. This experiment may
employs electron dephased rotational Hahn-echoes and adiabatic
inversions of the electron spin only made possible with frequency
agile gyrotron technology disclosed herein. FIG. 8B shows a scheme
in which frequency modulated MW power (labeled DNP) transfers EPR
polarization efficiently to nuclei, followed by adiabatic EPR
inversions to measure long-range electron-nuclear distances and
hyperfine decoupling during NMR acquisition.
[0046] Similar to heteronuclear distance measurements, the
dephasing of rotational Hahn-echoes may be monitored as a function
of the EPR adiabatic inversion placement in the MAS rotor cycle.
FIG. 9A shows strong hyperfine couplings may enable long-range
distance measurements (see top graph) compared to weak
.sup.13C-.sup.13C couplings (see bottom graph). FIG. 9B is a curve
fit to precise long-range distances yielded by the dephasing of
rotational Hahn-echoes with rotor synchronized adiabatic EPR
inversions as disclosed herein. As seen in FIG. 9C, similar curves
may be fit to yield precise .sup.13C-.sup.13C distances, but only
out to about 5 .ANG.. Due to the strength of the hyperfine
interaction, it may be possible to measure the 15-20 .ANG.
distances as indicated in FIG. 9A. In an aspect, long-range
distances may be measured between rigid nitroxide labels and
.sup.13C labels both on residues in binding pockets and on ligands
with a .+-.0.2 .ANG. precision.
[0047] In an aspect, the transverse electron relaxation may be
extended to enable adiabatic EPR inversions. This may be
accomplished with deuteration of residues near the nitroxide moiety
and by cooling the sample as cold as possible. In one aspect, the
sample may be cooled to a temperature less than about 27
Kelvin.
Microwave Frequency Modulation for Broad-banded Electron-Nuclear
Decoupling
[0048] Extending the decoupling strategies discussed herein above
to DNP using nitroxide radicals and the 3-spin Cross Effect
mechanism may require a frequency modulation of the microwaves
across the entire broad EPR lineshape. Such modulation of the
microwave frequency from about 197.0 to 198.3 GHz (see shading in
top of FIG. 2A) may be accomplished by modulating the operating
voltage of the gyrotron by .+-.1.4 kV. Continuous waveforms applied
in NMR may have similar analogies to EPR and DNP transitions.
Analogous to the first heteronuclear decoupling experiments, a
random modulation of MW frequency over the lineshape might better
average out electron-nuclear couplings.
Magic Angle Spinning (MAS) Solid State NMR
[0049] The NMR Hamiltonian contains anisotropic terms such as
dipolar interactions and chemical shift anisotropy that can lead to
short relaxation times and line broadening in NMR spectra of solid
state samples. However, a factor of (3 cos.sup.2.theta.-1) in these
Hamiltonians allows effectively averaging weaker anisotropic
interactions to zero (3 cos.sup.254.7.degree.-1=0) with mechanical
rotation of the sample at 54.7.degree. (the magic angle) with
respect to the magnetic field (FIG. 7A), resulting in narrow NMR
resonances.
Electron Dephased Rotational Echo DOuble Resonance (EDREDOR)
[0050] Rotational Echo DOuble Resonance (REDOR), correlates the
amount of dephasing during a spin-echo to distances between nuclear
spin pairs--the closer the "dephasing" spin is to the "observed"
spin, the stronger the dephasing. Similarly, spins with larger
gyromagnetic ratios yield more dephasing, enabling longer distance
measurements up to about 12 A for .sup.19F-.sup.13C spin pairs.
Electron spins have magnetic moments about 660 times larger than
.sup.19F nuclear spins. These strong electron spins may be used to
measure electron-nuclear distances on a protein out to about 50
.ANG.. The pulse sequence for such an Electron Dephased REDOR
(ED-REDOR) experiment is shown in FIG. 3, which includes an
adiabatic inversion of the electron spin only made possible with
frequency agile gyrotron technology. In typical REDOR experiments,
.pi. pulses refocus magnetization, but in the experiment
illustrated in FIG. 3, an adiabatic inversion on the electron spin
interferes refocusing of the spin echo. The extent to which the
magnetization is dephased directly encodes the electron-nuclear
distance, and also the orientation of the dipolar vector. Similar
to REDOR, ED-REDOR may also allow the measurement of the
orientation of the electron-nuclear dipolar vector in addition to
the measurement of the electron-nuclear distance.
[0051] Similar experiments exist in EPR, such as ENDOR (Electron
Nuclear Double Resonance). EDREDOR is different in a few very
important ways. Primarily, ED-REDOR is conducted during a MAS
experiment that yields high resolution NMR spectra. The
disadvantage to the MAS experiments is the lack of an EPR resonant
structure--this is why frequency agile gyrotrons are so critical.
Their high power levels compensate for the lack of EPR resonant
structure, enabling an adiabatic inversion of the electron spins.
Also, ENDOR is EPR detected, which limits the range of distance
measurements to about 15 .ANG.. ED-REDOR is also similar to solid
state NMR structural measurements with paramagnetic relaxation
effects. However, ED-REDOR has a 1/r.sup.3 distance dependence
versus the 1/r.sup.6 dependence of EPRs, making it possible to
measure out to about 50 .ANG. rather than 15 .ANG..
[0052] One of the challenges to implementing ED-REDOR is extending
the transverse electron relaxation to enable the adiabatic
inversion, and longitudinal electron relaxation time to allow for
the long mixing times required to measure long distances. This will
be accomplished by cooling the sample to as low a temperature as
possible. In one aspect, the sample may be cooled to a temperature
below about 20 Kelvin.
Polarizing agents and EPR spin labeled proteins
[0053] The stable organic radicals and EPR transition metals to be
used for Cross Effect DNP, Solid Effect DNP, and electron-nuclear
distance and dipolar orientation measurements have not previously
been used for electron-decoupling or installing radicals on
proteins for use with DNP because high power frequency agile
gyrotrons and electron nuclear decoupling are needed. TOTAPOL (FIG.
13A) is an exogenous biradical polarizing agent comprised of two
nitroxide moieties tethered together. The broad EPR lineshape and
strong electron-electron dipolar coupling enabled by the frequency
agile gyrotron is combination with TOTAPOL may yield efficient DNP
with the Cross Effect. TOTAPOL is the currently the most common
polarization agent used in magic angle spinning DNP, and may be
used with in vivo studies. The broadbanded microwave irradiation
from the frequency agile gyrotron as disclosed, which may cover the
entire nitroxide lineshape, may enable powerful electron nuclear
decoupling and distance measurements. In an aspect, more narrow
line EPR moieties may also be used.
[0054] Narrow line EPR resonances like that in water-soluble BDPA
(FIG. 13B) and gadolinium (FIG. 13C), are well-suited for Solid
Effect DNP, especially when high electron nutation frequencies are
available. The DNP enhancements from the Solid Effect still
increase linearly with respect to microwave power, even when gB1=3
MHz. The 100 Watt power levels achieved by the frequency agile
gyrotron as disclosed herein and EPR resonant structures may yield
very high electron nutation frequencies in the range of about 5 MHz
to about 30 MHz, or even higher.
[0055] Gadolinium is well-suited for Solid Effect DNP and electron
nuclear decoupling for EPR spin labels on proteins. Although
gadolinium has been used as a polarizing agent for DNP, and also
been installed on proteins to make electron-electron measurements,
performing DNP on a spin labeled protein has proven challenging.
The narrow central EPR transition linewidth of gadolinium is
dominated by isotropic zero-field splitting, which may simplify the
implementation and data interpretation of electron nuclear distance
measurements. However, the electron spin relaxation times of
gadolinium are much shorter than nitroxides. Such fast relaxation
makes it more challenging to manipulate these spins, especially to
measure electron nuclear distances. Extreme sample cooling, in one
aspect to temperatures of below about 15 Kelvin may enable combined
gadolinium EPR and NMR.
[0056] TOAC (FIG. 13C) is a nitroxide amino acid incorporated with
solid phase peptide synthesis (SPPS), its rigid conformation will
lead to higher precision electronuclear measurements. MTSSL (FIG.
13C) may be installed onto cysteine residues, and may be used
especially on protein GB1. As discussed previously, electron
nuclear decoupling and collapsing the spin diffusion barrier may
permit DNP at higher temperatures (about 220-273 K), where
exquisite resolution in solid state NMR spectra of microcrystalline
GB1 has been demonstrated.
Gyrotron and DNP Probe
[0057] A gyrotron, generally disclosed herein as 100 in FIG. 1, may
be frequency agile and have a wide instantaneous bandwidth. The
frequency agile gyrotron 100 may include an output window 104, an
interaction cavity 106, and an electron gun 108. An electron beam
102 may extend through the length of the frequency agile
gyrotron.
[0058] The frequency agile gyrotron 100 may be operatively
connected to a NMR DNP probe 400. FIG. 4A is a schematic of a probe
head for 300 MHz/200 GHz magic angle spinning DNP including a
microwave waveguide 404 and vacuum jacketed cryogen lines 408, 410,
which may be connected from the top of the magnet. FIG. 4B is a
cross section showing the large sample volume 412 (about 250 .mu.L)
for in vivo and extremely high sensitivity in vitro studies. In an
aspect, the DNP NMR probe may include a quadruple resonant NMR coil
414. FIG. 4C shows a DNP probe that includes a helium variable
temperature line that cools samples to between about 25 Kelvin and
about 27 Kelvin, a high-performance RF transmission line circuit
416, and an efficient microwave transmission line. FIG. 4D
illustrates a vacuum jacketed Dewar 428 for 89 mm bore magnets with
connection ports 430 to the top of the magnet. FIG. 4E is a
schematic showing the cold helium gas flow along to rotor surface
for efficient heat-exchange, then out exhaust ports 402 that
establish a heat shield. The DNP probe may further include a drive
cup (turbine) 418 and at least one bearing 420. FIG. 4F is an
illustration of 200 GHz radiation in a DNP cavity including a
microcoil 422 for about 1 .mu.L samples 426. The microcoil 422 may
provide improved EPR performance. Cross sections of the inductively
coupled RF coils 422 and concave GHz mirrors 424 may generate about
>1 MHz nutation fields on nuclear spin RF channels and >10
MHz nutation of electron spins resonating at about 200 GHz. FIG. 4G
is a High Frequency Structure Simulation (HFSS) at 250 GHz of the
sample chamber on the probe in FIG. 4C. FIG. 7 is a simulation of a
microwave electromagnetic field of the DNP sample chamber
412/426.
[0059] FIG. 10A is a schematic of a Fabry-Perot EPR resonator 1000
for magic angle spinning DNP. The resonator 1000 may include a
magic angle spinning N.sub.2 (g) bearing 1008 and a sapphire rotor
1014. FIG. 10A illustrates a Teflon lens 1002 that focuses the
microwave power from the gyrotron 100 to the sample 1016 using a
corrugated waveguide 1004 in between turns of the radio-frequency
coil 1006. An adjustable copper mirror 1010 may excite the cavity
mode. FIG. 10B illustrates the dielectric constants (impedance) of
the sample 1016 and sapphire rotor 1014 are matched with
anti-reflective THz coatings 1012 to reduce loss. FIG. 10C
illustrates the cavity mode establishes a microwave field over the
sample 1016.
[0060] The broadband microwave irradiation generated from the
frequency agile gyrotron 100 may allow significantly more control
over the DNP Hamiltonian. Beneficial hyperfine interactions may be
able to be turned on and detrimental ones turned off to obtain
significantly more sensitive and precise biomolecular structural
refinement. For instance, electron decoupling may be implemented,
which is analogous to proton decoupling and electron-nuclear
distance measurements that are analogous to nuclear-nuclear
measurements.
[0061] The electron beam 102 ejected from cathodes in the frequency
agile gyrotron 100 may generate microwave power that can interact
with EPR spins at about 197 GHz. In the frequency agile gyrotron
100, the electron acceleration voltage between the cathode and
anode determines the microwave output and may be changed quickly to
permit electron nuclear decoupling (FIGS. 8A, 8B, and 8C). For
instance, a frequency jump of about 300 MHz to switch from the DNP
transfer condition to direct irradiation of the EPR resonances for
decoupling may be achieved by decreasing the operating voltage of
the gyrotron by about 670 V.
[0062] The about 5 Kelvin helium gas flow indicated by the arrows
in FIG. 12A may effectively cool the sample 1208 while a bearing
420 fed with cold nitrogen gas (about 60 K) provides stable magic
angle spinning. In an aspect, the sample 1208 may be at about 20
Kelvin. This design employs helium exhaust ports 402 (see FIG. 12B)
that force the helium to be in extended contact with the NMR rotor
to increase heat exchange efficiency, and also to cool heat shields
surrounding the sample 1208. The system may further include ports
1222 (see FIG. 12B) for sample ejection, MAS bearing, MAS drive,
helium cooling, and 200 GHz channel. The drive-tip 1204 (see FIG.
12A) and pressurized N.sub.2 (g) turbine power may provide the
centrifugal force to compress helium. The arrows in FIG. 12A
indicate helium flow and expansive cooling of the sample 1208
followed by compression. The helium may flow to a miniature heat
exchanger where heat is exchanged with a second closed helium loop
cooled by a cryogenic refrigerator. In an aspect, the helium may
flow from a refrigerator 1214, to a heat exchanger for re-cooling
1216, from the heat exchanger 1210, and return to the refrigerator
for re-cooling 1212. A helium propeller 1218 and a helium impeller
1220 may be used to move the helium through the system, as
illustrated in FIG. 12A.
[0063] In the frequency agile gyrotron as disclosed herein, the
operational voltage determines the microwave output frequency. In
an aspect, the voltage-tunable gyrotron may change the frequency by
about 1 GHz in about 1 second. The frequency agile gyrotron may
allow for much faster voltage control and adiabatic inversions
through the about 1 GHz nitroxide lineshape in about 2 .mu.s. In an
aspect, the gyrotron may operate in a "chaotic" mode of operation
for hyperfine decoupling. The physics of the interaction of the
electron beam with the interaction cavity is shown in FIG. 8C. The
gyrotron quickly switches between axial modes resulting in short
(<1 ns) pseudo-random pulses that are excellent for hyperfine
decoupling. In an aspect, the instantaneous bandwidth of the 200
picosecond pulse in FIG. 8D may be about 4 GHz and may completely
cover the 1 GHz nitroxide lineshape.
[0064] To perform NMR experiments relevant to biomolecular
structure determination with DNP, probe instrumentation may perform
a diverse set of tasks including; control of nuclear spins with
efficient multiresonant RF circuits, control of EPR spins with GHz
irradiation using waveguide and quasioptics, and cryogenic cooling
with stable about 5 KHz to about 8 KHz magic angle spinning.
Spinning at the magic angle (54.7.degree.) averages anisotropic
interactions in the Hamiltonian and results in narrow NMR
resonances and resolved spectra. The high-performance quadruple
resonance NMR DNP probes must retain the capability to implement
all of the homo and heteronuclear polarization transfer and
decoupling schemes that are integral to solid-state NMR
spectroscopists.
[0065] RF transmission line circuits may generate about 83 KHz
nutation frequencies on multiple RF channels and may be employed
for rotors up to 7 mm diameter with sample volumes of up to 250
.mu.L (FIG. 4A, 4B, 4E, 4G). Most DNP probes typically have about
30 .mu.L sample volumes. The new DNP probe accommodating larger
rotors will enable improved sensitivity for in vivo samples.
[0066] Sometimes it may be prohibitively difficult to make such
large sample volumes of isotopically labeled protein and drugs.
Therefore, inductively coupled microcoils that house sample volumes
of about 1 .mu.L may be incorporated into the DNP probes in an
aspect. In addition to achieving excellent filling factors and
sensitivity with 1 .mu.L sample volumes, there are many additional
advantages to microcoils from a RF and GHz perspective.
[0067] Challenges to achieving high quality EPR resonators in MAS
include coupling the microwave power efficiently into the sample,
and addressing losses of microwave power from the lossy sample. The
system in FIG. 4F accomplishes both of these. Concave mirrors 424
placed in the rotor itself and opposite the waveguide aperture
focus the microwave power into the sample 426. A 1 .mu.L sample,
although lossy, is small compared to the large size of the EPR
resonant structure, leading to high quality factors. Together with
the high power levels achieved by the frequency agile gyrotrons
disclosed herein, EPR quality factors of 5 to 80, corresponding to
electron nutation frequencies of 8-32 MHz may be achieved. Such
strong control of the electron spins may lead to tremendous
advancements in magnetic resonance methods.
[0068] Microcoils may also generate very high nutation frequencies
of the nuclear spins. With the high power amplifiers already in
place on the spectrometer, high efficiency RF transmission line
circuits, and microcoils, 0.1-1 MHz nutation frequencies may be
generated on .sup.1H, .sup.19F, .sup.31P, .sup.13C, .sup.15N,
.sup.2H; all simultaneously. Not all of the NMR pulse sequences may
make use of all of these channels in the same experiment. In an
aspect, the .sup.1H channel may be used for cross polarization and
.sup.1H decoupling and the .sup.19F and .sup.2H channels may
manipulate isotopically labeled spins on bryostatin. In addition,
the .sup.31P channel may control .sup.31P spins on phospholipid
head groups and phosphorylated tyrosine residues in the active site
of a protein. Correlations between .sup.13C and .sup.15N on
uniformly labeled proteins between the .sup.19F, .sup.2H, and
.sup.31P spins may yield not only distance constraints, but also,
spectral filtering to clear-up spectral congestion. For
site-resolved spectra of fully labeled proteins of >400 amino
acids using .sup.13C and .sup.15N, uniquely resolved spins and
advance probe technology as described herein may be utilized.
Building instrumentation that can manipulate seven types of spins
(including the electron spins) simultaneously represents a huge
leap forward in innovation from typical solid state NMR probes that
are triple resonance.
[0069] Among the challenges to achieving high quality EPR
resonators in MAS experiments of >5 Tesla include coupling the
microwave power efficiently into the sample, and overcoming losses
of microwave power in the resonator. The Fabry-Perot resonance
structure 1000 shown in FIGS. 10A, 10B, and 10C accomplishes both
of these objectives. An adjustable Teflon lens 1002 couples the 200
GHz microwave power 1004 from the gyrotron 100 efficiently into the
EPR resonator 1000 through a split radio-frequency coil 1006
design. An adjustable concave copper mirror 1010 optimizes the
boundary condition to excite a strong cavity mode (FIG. 10C).
Anti-reflective coatings 1012, and a sapphire sample rotor 1014
minimize loss of power when the microwaves pass through materials
with different dielectric constants (FIG. 10B). A High Frequency
Structure Simulator may be used to calculate the propagation of the
microwaves and optimize the materials and geometry in the EPR
resonator. High quality factors (Q.about.100) can be achieved
because the lossy sample 1016 is small compared to the size of the
EPR resonant structure 1000. There are no EPR resonators currently
available for high-frequency MAS DNP; the EPR quality factor of
existing instrumentation is Q.about.1.
Efficient Helium Cooled Magic Angle Spinning
[0070] Enhanced sensitivity in magnetic resonance is available at
cryogenic temperature due to a 1/T dependence of the spin
polarization (see Eqn. (I)). Electron and nuclear spin relaxation
times also increase drastically at lower temperatures, permitting
efficient transfers of the enhanced electron polarization to
nuclear spins. Consequently, most MAS DNP experiments are performed
at 80-100 Kelvin where inexpensive N.sub.2 (g) can be used both to
spin the NMR sample and to provide cooling. There are some initial
studies that use about 6 L/hr of liquid helium to cool samples to
about 25 K, but are encumbered by the ever-increasing high cost of
helium. Current efforts to recycle the helium entail a tremendous
investment in infrastructure and laboratory space (see FIG. 11,
bottom).
[0071] The frequency agile gyrotron system may further include a
miniature helium recirculation system shown in FIG. 11 (top) and
FIGS. 12A and 12B. A centrifugal compressor 1202 mounted on the
rotor harnesses the rotational kinetic energy afforded by the
drive-tip turbine 1204 (FIG. 12A) and about 8 kHz sample rotation.
The helium may circulate between a miniature heat-exchanger 1226
and the sample 1208 (see FIGS. 12A and 12B). The helium flowing
over the rotor may be cooled to about 5 K, but the sample may be
about 25 K due to heat conduction along the rotor and microwave
heating.
[0072] The geometry of the rotor fins and compression manifold may
yield the flow pattern shown in FIG. 12. At field strengths of
about 7 Tesla, a spin at about 8 KHz may yield narrow linewidths in
the MAS NMR spectra. Vacuum jacketed insulation may be critical to
separate the helium loop from the sample chamber 1208 maintained at
90 K with N.sub.2 (g).
[0073] Previous experiments below 10 K required tens of liters of
helium an hour. There has been a renewed interest in cold helium
spinning in solid-state NMR the last decade, and the drive has been
to make the cooling more efficient. The length of the rotor may be
extended and tight disks (baffles) used to isolate the cold sample
region in the center of the rotor. The helium gas flow indicated by
the blue arrows in FIG. 4E may be used. This design employs helium
exhaust ports 402 that force the helium to be in contact with the
rotor much longer to increase heat exchange efficiency, and also to
cool the disks surrounding the sample to establish a heat shield.
These improvements, in addition to using about 100 Kelvin MAS
spinning gases, may allow sample temperatures of less than about 20
Kelvin with minimal helium consumption. In an aspect, the helium
consumption may be less than about 1 L/hr.
Spectrometer Control of the Gyrotron Frequency
[0074] In tunable gyrotrons, the acceleration voltage between the
cathode and anode in the magnetron injection gun dictates the
microwave output frequency (see FIG. 9B, right). Control of the
voltage (and frequency) of the gyrotron by the NMR spectrometer
enables seamless integration between the NMR and EPR channels in
DNP experiments. The TECMAG spectrometer may have extra arbitrary
waveform generators with a 10 ns step size. Thus, implementing the
voltage and frequency agility schemes shown in FIG. 6 may be
accomplished from the integrated DNP spectrometer console. Voltage
output channels from the spectrometer may be amplified (and
optically isolated), added together in a biasing circuit, and
connected to the gyrotron anode. The voltage on the anode controls
the electric potential (see FIG. 6, right), thus tuning the
velocity of the electron beam and the microwave frequency
output.
Protein Sample Preparation for NMR
[0075] Previously, dozens of milligrams of protein for atomic level
structural biology was required. In an aspect, the sample size for
in vitro samples may be decreased. With the microcoil
instrumentation and DNP sensitivity described previously herein,
there may be excellent sensitivity with about 1 .mu.L samples. For
example, with about 200 .mu.g of protein (most of the volume is
taken up by lipids), a full-length protein in eukaryotic cells may
be expressed and the functional kinase may be purified without need
for optimizing yields at every step of the protocol. Similarly, 200
.mu.g of a protein from solid phase peptide synthesis may enable
incorporation of selectively isotopically labeled residues and EPR
tags. At the same time, it will be much easier to provide about 30
.mu.g of sample versus the 4 mg currently needed.
[0076] In vitro measurements may benefit from DNP sensitivity. For
instance the sensitivity may be leveraged to determine drug and
protein confirmations present at a minute fraction of the
cryogenically trapped ensemble. Often these thermodynamically less
favored states are critically the most important
structures--excursions in the energy landscape that result in drug
binding, dissociation, and catalysis.
EXAMPLES
Prophetic Example 1
[0077] FIG. 4B shows the chemical structure
1,3-bisdiphenylene-2-2-phenylallyl (water soluble BDPA), the
exogenous stable organic radical that may be a source of the
enhanced magnetic resonance polarization (sensitivity). FIG. 2A
shows the electron paramagnetic (EPR) spectra of nitroxide and BDPA
radicals in red. The enhancement profiles show the level of
polarization enhancement obtained on the nuclear spins by sweeping
the microwave irradiation frequency (or NMR magnet field strength)
through the DNP matching conditions. For the narrow line BDPA
radical, the Solid Effect, a two-spin DNP mechanism is active when
the microwave irradiation frequency is 300 MHz (the proton nuclear
Larmor frequency) away from the EPR resonance. Protons are
polarized from the Solid Effect when the gyrotron is set to 197.0
GHz and the EPR resonance is at 197.3 GHz.
[0078] An in vitro NMR sample will contain about 200 micrograms of
isotopically labeled bryostatin and also PKC C1b domain,
phosphatidyl serine lipids, BDPA DNP polarizing agent, and a
cryoprotecting matrix of glycerol. The sample will be loaded into a
rotor for magic angle spinning (MAS). For in vivo ligand structural
determination, about 400 mg of human cells (HeLa or similar) will
be treated with isotopically labeled bryostatin, spun down, and
then resuspended in a cryoprotecting glycerol matrix with dissolved
DNP polarizing agent, before being centrifuged into a MAS NMR
rotor.
[0079] Simultaneous radio frequency irradiation resonant with
.sup.1H, .sup.2H, .sup.31P, .sup.13C, and .sup.15N spins from a
custom designed NMR radio frequency circuit may yield sufficient
control of the nuclear spins to attenuate elements in the NMR
Hamiltonian that lead to line broadening, while also permitting the
measurement to sub-angstrom precision between .sup.13C, .sup.15N,
.sup.2H isotopic labels on bryostatin and .sup.31P spins on the
phospholipid head groups.
[0080] With the microcoil instrumentation and DNP sensitivity
described previously herein, there may be excellent sensitivity
with about 1 .mu.L samples. For example, with about 200 .mu.g of
protein (most of the volume is taken up by lipids), a full-length
protein in eukaryotic cells may be expressed and the functional
kinase may be purified without spending a lot of time trying to
optimize yields at every step of the protocol. Similarly, in a
cost-effective manner, 200 .mu.g PKC C1b domain from solid phase
peptide synthesis will permit incorporation of selectively
isotopically labeled residues and EPR tags. At the same time, it
may be easier to provide about 30 .mu.g of isotopically labeled
bryostatin analogs versus the 4 mg currently needed.
[0081] In vivo NMR spectroscopy may ensure that the PKC is bound to
endogenous lipids along with all of the co-factors, anchoring
proteins, scaffold proteins, and other macromolecules present in
the membrane that play a role in regulation. For these in vivo
experiments, large 250 .mu.L sample volumes may be used--it is not
difficult to culture cells and spin them down in a centrifuge to
get about 200 mg quantities; the tough part is always the
purification, refolding, and reconstituting into lipids. The in
vivo spectroscopy may be extended to primary cells and determine
the structures of bryostatin and phorbol in diseased tissue.
[0082] Long-range distances will be measured between rigid
nitroxide labels on the C1b domain and .sup.13C labels both on
residues in the binding pockets and on ligands with a .+-.0.2 .ANG.
precision.
[0083] The examples described herein are included to demonstrate
preferred embodiments of the invention. It should be appreciated by
those of skill in the art that the techniques disclosed in the
examples included herein represent techniques discovered by the
inventors to function well in the practice of the invention, and
thus can be considered to constitute preferred modes for its
practice. However, those of skill in the art should, in light of
the present disclosure, appreciate that many changes can be made in
the specific embodiments which are disclosed and still obtain a
like or similar result without departing from the spirit and scope
of the invention.
* * * * *